Protein Chemistry. Structure, Properties, Research Methods - Shendryk, A.N. 2022

Methods for Experimental Investigation of Protein Structure
Electron Impact Mass Spectrometry
Sample Preparation Methods for Mass Spectrometry

A protein molecule or its fragment (peptide) typically contains a variety of polar functional groups. While this enhances their Water solubility, it renders them practically insoluble in organic Solvents and significantly lowers their volatility. For a long time, this hindered The Development of a reliable methodology for the mass spectrometric analysis of Peptides. Overall, it took more than a decade to establish this approach. It is based on advanced techniques for converting water-soluble peptides into chloroform-soluble, volatile derivatives. One of the most versatile Methods FOR PREPARING volatile peptide derivatives for mass spectrometric analysis allows modification directly within a mixture containing 5-100 nol of each component. This modification scheme involves converting peptides into N-acetyl-N,O,S-permethyl derivatives. Prior to this chemical modification, Arginine-containing compounds are converted into ornithyl peptides. To identify arginine-containing peptides in the sample, a portion of the mixture undergoes preliminary Amino acid analysis or analytical Electrophoresis followed by staining.

Acetylation.

Treating the sample with acetic anhydride at room Temperature for one minute protects only the N-terminal amino group of the peptide, whereas Modification of the ε-amino groups is achieved within 3 hours. To reduce the acetylation time for both α- and ε-amino groups, the reaction is carried out in the presence of bases.

Permethylation.

Acetyl peptides are methylated as follows. First, the base—methylsulfinyl carbanion—is prepared. To do this, sodium hydride is added to 3-4 ml of dimethyl sulfoxide, and the reaction mixture is incubated for 20 min at 90°C. The completion of the reaction is indicated by the appearance of an orange-brown color and the cessation of hydrogen evolution. Next, the prepared methylsulfinyl carbanion solution is added in excess to the peptide dissolved in dimethyl sulfoxide. After one minute, methyl iodide is added, and methylation is carried out for 70 s. Once this time has elapsed, the reaction is quenched by adding water. The resulting permethylated acetyl peptides are immediately extracted from the mixture with chloroform.

Hydrazinolysis of Arginine Residues.

50 µl of an aqueous hydrazine hydrate solution (1:1) is added to 50-100 nmol of the peptide and incubated for 12 min at 80°C. Afterward, the reaction mixture is cooled, 50 µl of water is added, frozen using liquid nitrogen or an ethanol-dry ice (CO₂) bath, and vacuumed until the water is completely removed. The sample is then slowly thawed to room temperature.

A generalized flowchart of the entire peptide modification process prior to Electron Impact Mass Spectrometry analysis

is given below.

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Scheme of acetylation and exhaustive methylation of peptides for mass spectrometry

Conversion of arginine residues to Ornithine residues